Human immunodeficiency virus type 1 nucleic acids devoid of long terminal repeats capable of encoding for non-infectious, immunogenic, retrovirus-like particles

ABSTRACT

Non-infectious, retrovirus-like particles contain mutations to reduce gag-dependent RNA-packaging of the gag gene product, eliminate reverse transcriptase activity of the pol gene product, eliminate integrase activity of the pol gene product and eliminate RNase H activity of the pol gene product through genetic manipulation of the gag and pol genes. The corresponding nucleic acid molecules are described. The non-infectious, retrovirus-like particles have utility in diagnosis.

This is a continuation of application Ser. No. 08/292,967 filed Aug. 22,1994, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with retrovirus-like particles (sometimes termedpseudovirions), made non-infectious by a plurality of mutations.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus is a human retrovirus and is theetiological agent of acquired immunodeficiency syndrome (AIDS). SinceAIDS was first reported in the U.S. in 1981, more than 194,000 peoplehave died of AIDS and over 330,000 cases of HIV infection have beenreported in the U.S. alone. Worldwide, it is estimated that more than 17million people have been infected with HIV.

More than 100 AIDS-related medicines are in human clinical trials orawaiting FDA approval but there is currently no cure for the disease.

There is, therefore, a clear need for immunogenic preparations useful asvaccine candidates, as antigens in diagnostic assays and kits and forthe generation of immunological reagents for diagnosis of HIV and otherretroviral disease and infection.

Particular prior art immunogenic preparations include non-infectious,non-replicating HIV-like particles. Thus PCT applications WO 93/20220published Oct. 14, 1993 and WO 91/05860 published May 2, 1990 (WhiteheadInstitute for Biomedical Research), teach constructs comprising HIVgenomes having an alteration in a nucleotide sequence which is criticalfor genomic RNA packaging, and the production of non-infectiousimmunogenic HIV particles produced by expression of these constructs inmammalian cells.

PCT application WO 91/07425 published May 30, 1991 (Oncogen LimitedPartnership) teaches non-replicating retroviral particles produced byco-expression of mature retroviral core and envelope structuralproteins, such that the expressed retroviral proteins assemble intobudding retroviral particles. A particular non-replicating HIV-1 likeparticle was made by coinfecting mammalian host cells with a recombinantvaccinia virus carrying the HIV-1 gag and protease genes and arecombinant vaccinia virus carrying the HIV-1 env gene.

In published PCT application WO 91/05864 in the name of the assigneehereof (which is incorporated herein by reference thereto), there aredescribed particular non-infectious, non-replicating retrovirus-likeparticles containing at least gag, pol and env proteins in their naturalconformation and encoded by a modified retroviral genome deficient inlong terminal repeats and containing gag, pol and env genes in theirnatural genomic arrangement.

Virions of HIV comprise two copies of the single-stranded RNA genomeenclosed within a capsid. After penetration into a susceptible hostcell, the HIV genome is copied by the viral reverse transcriptase intosingle-stranded DNA that is thought to be translocated into the nucleus,wherein a cellular DNA polymerase synthesizes the second DNA strand. Thedouble-stranded copy is then integrated, at random, into one of the hostchromosomes, resulting in a duplication of a region of the viral genomeat the extremities of the genome. The long-terminal repeat (LTR) of theintegrated provirus is recognized by a cellular RNA polymerase and thetranscribed RNA is translated to give rise to viral proteins. The RNAtranscripts can also be packaged into new virions that leave the cell bya process of budding.

The HIV genome encodes at least nine different proteins. The three majorgenes, gag, pol and env are common to all retroviruses and encode virionproteins.

The differential expression of these genes is achieved through a complexpattern of processing of the primary precursor transcript. Only the GAGand POL proteins are produced from the unspliced mRNA corresponding tothe genomic RNA of the virion. The ENV protein is translated from a mRNAspecies that has undergone a single splicing event to delete the gag andpol coding sequences, and other proteins are produced from mRNA speciesthat are spliced several times. The general structure of HIV is reviewedby Kieny et al (ref. 8).

Thus, it may be advantageous under particular circumstances to produceretrovirus-like particles (and in particular HIV-like particles) bymutating other portions of the HIV genome contributing to infectivityand replication of the virus. Such modifications may be modifications ofthe gag and pol gene products.

There is currently no vaccine nor effective treatment for AIDS.Heat-inactivated anti-HIV antiserum obtained from HIV-infected peopleand inactivated HIV are currently commercially available as componentsof many diagnostic methods. For safety, ease of handling, shipping,storage and use, it may be preferable to replace such antigen andheat-inactivated antisera by non-infectious HIV-like particles andantisera generated by immunization with non-infectious HIV-likeparticles as described above and particularly in WO 91/05864.Furthermore, antisera generated by immunization with thesenon-infectious HIV particles do not require heat inactivation to removeinfectious HIV. The HIV-like particles described in WO 91/05864 areentirely deficient in replication and infection. However, because of theseriousness of HIV infection, it may be desirable under certaincircumstances to provide retrovirus-like particles deficient in aplurality of elements required for infectivity and/or replication of HIVbut dispensible for virus-like particle formation. Furthermore, sinceprior art HIV-like particles contain many of the HIV proteins insubstantially their natural conformations, a host immunized therewithmay mount an immune response immunologically indistinguishable frominfection by HIV and it may be desirable to be able to distinguishbetween inactivated HIV and non-infectious, non-replicating HIVparticles and antisera generated by virulent HIV and non-infectious,non-replicating HIV-like particles. Thus, in the development of AIDSvaccine candidates, immunogenic preparations and diagnostic methods andkits, it would be useful to provide an HIV-like particle deficient in aplurality of elements required for infectivity and/or replication andoptionally immunologically or otherwise distinguishable from virulentHIV.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision ofretrovirus-like particles made non-infectious by a plurality ofmutations.

Accordingly, in one aspect of the invention there is provided anon-infectious immunogenic, retrovirus-like particle comprising, in anassembly, gag, pol and env gene products, wherein at least onemodification has been made to the pol and/or gag gene product, to effectat least one of the following :

(a) reduce gag-dependent RNA packaging of the gag gene product;

(b) substantially eliminate reverse transcriptase activity of the polgene product;

(c) substantially eliminate integrase activity of the pol gene product;and

(d) substantially eliminate RNase H activity of the pol gene product.The reduction in gag dependent RNA packaging may be effected byreplacing or deleting at least one amino acid residue contributing togag-dependent RNA packaging in the gag gene product. In an illustrativeembodiment, the at least one amino acid may be contained within aminoacids Cys³⁹² to Cys³⁹⁵ of the gag gene product of HIV-1 LAI isolate orthe corresponding region of other retroviral gag gene products andCys³⁹² and/or Cys³⁹⁵ or both cysteines may be replaced by serineresidues.

In one specific illustrative embodiment of the invention, thesubstantial elimination of reverse transcriptase activity of the solgene product may be effected by deletion of at least a portion thereofcontributing to reverse transcriptase activity. The at least a portionof the pol gene product may be contained between amino acids Pro¹⁶⁸ andLeu⁷²⁷ of the pol gene product of HIV-1 LAI isolate or the correspondingregion of other retroviral pol gene products. The substantialelimination of integrase activity of the pol gene product may beeffected by deletion of at least a portion thereof contributing tointegrase activity and the at least a portion of the pol gene productmay be contained between amino acids Phe⁷²⁸ and Asp¹⁰¹⁶ of the pol geneproduct of HIV-LAI isolate or the corresponding region of otherretroviral pol gene products.

The substantial elimination of RNase H activity of the pol gene productmay be effected by deletion of at least a portion thereof contributingto RNase H activity.

In a particular embodiment of this aspect of the invention substantialelimination of reverse transcriptase, integrase and RNase H activitiesmay be simultaneously effected by deleting a portion of the pol geneproduct corresponding to amino acids Pro¹⁹² to Trp⁸³⁵ of HIV-1 LAIisolate, or the corresponding region of other retroviral pol geneproducts.

In a further aspect of the invention, the non-infectious retrovirus-likeparticles of the invention may additionally comprise at least onenon-retroviral antigenic marker. The incorporation of antigenic markersinto non-infectious retrovirus-like particles is described in ourcopending U.S. Pat. application Ser. No. 08/290,105 filed Aug. 15, 1994,the disclosure of which is incorporated herein by reference. The atleast one antigenic marker may be contained within the gag gene productto form a hybrid gag gene product having the particle-formingcharacteristics of unmodified gag gene product. In a particularembodiment, the at least one antigenic marker may be inserted into aninsertion site of the gag gene product at an antigenically-activeinsertion site and the insertion site may be located between amino acidresidues 210 and 211 of the gag gene product of the HIV-1 LAI isolate orthe corresponding location of other retroviral gag gene products. The atleast one antigenic marker may comprise from 1 to 4 tandem copies of theamino acid sequence AFDTRNRIIEVEN (SEQ ID NO: 1) or a portion, variationor mutant thereof capable of eliciting antibodies that recognize thesequence AFDTRNRIIEVEN.

The marker sequence also may be provided by deleting or preventingproduction of an amino acid sequence that corresponds to an epitope of aretroviral protein. Such epitope may comprise the immunodominant epitopeof gp41, which provides endogenous anchoring function. When suchendogenous anchoring function is removed in this way, the anchoringfunction is provided by a different antigenic anchor sequence.

In a further particular embodiment of this aspect of the invention, theenv gene product of the retrovirus-like particles as provided herein maybe a modified env gene product in which endogenous anchoring functionhas been replaced by a different antigenic anchor sequence opertivelyconnected to the env gene product to anchor the env gene product to theretrovirus-like particle and the anchor sequence may be inserted into aninsertion site of the env gene product adjacent to and upstream offunctional cleavage sites of the env gene product. The insertion sitemay be located between amino acid residues 507 and 508 of the env geneproduct of the HIV-1 LAI isolate or the corresponding location of otherretroviral env gene products. The anchor sequence may include an aminoacid sequence WILWISFAISCFLLCVVLLGFIMW (SEQ ID NO: 2) or a portion,variation or mutant thereof capable of eliciting antibodies thatrecognize the sequence WILWISFAISCFLLCVV LLGFIMW.

In yet another embodiment, the anchor sequence may include an amino acidsequence STVASSLALAIMIAGLSFWMCSNG SLQ (SEQ ID NO: 3) or a portion,variation or mutant thereof capable of eliciting antibodies thatrecognize the sequence STVASSLALAIMIAGLSFWMCSNGSLQ.

In another embodiment, the anchor sequence may include an amino acidsequence WILWISFAISCFLLCVVCWGSSCG PAKKATLGATFAFDSKEEWCREKKEQWE (SEQ IDNO: 4) or a portion, variation or mutant thereof capable of elicitingantibodies that recognize the sequenceWILWISFAISCFLLCVVCWGSSCGPAKKATLGATFAFDSKEEWCREKKEQWE.

The retrovirus-like particle generally is a human retrovirus-likeparticle, particularly derived from HIV-1, HIV-2, HTLV-1 or HTLV-2.Specifically, the human retrovirus may be HIV-1 and the env gene productmay be an LAI env gene product, an MN env gene product, an env geneproduct from a primary HIV-1 isolate, or an env gene productantigenically equivalent thereto.

The present invention also includes nucleic acid molecules encoding thenon-infectious, retrovirus-like particles of the invention. Accordingly,in another aspect of the invention, there is provided a nucleic acidmolecule encoding a non-infectious, immunogenic, retrovirus-likeparticle, comprising a modified retroviral genome deficient in longterminal repeats and containing gag, pol and env genes in their naturalgenomic arrangement and means for expression operatively connected tothe modified retroviral genome for production of gene products in cellsto produce non-infectious, immunogenic, retrovirus-like particlescomprising an assembly of gag, pol and env gene products, wherein atleast one codon in the gag or pol gene has been mutated to effect atleast one of the following:

(a) reduce gag-dependent RNA packaging activity of the gag gene product;

(b) substantially eliminate reverse transcriptase activity of the polgene product;

(c) substantially eliminate integrase activity of the pol gene product;and

(d) substantially eliminate RNase H activity of the pol gene product.The nucleic acid molecule may comprise a DNA molecule containing thecharacteristic genetic elements present in a SacI 678 to XhoI 8944fragment of the genome of the HIV-1 LAI isolate. The modified genomealso may be deficient in primer binding site and/or an RNA packagingsignal.

The reduction of gag-dependent RNA packaging may be effected bymutagenesis of a region thereof encoding at least one amino acidcontained with a region of the gag gene product corresponding to Cys³⁹²to Cys³⁹⁵ of the HIV-1 LAI isolate, or the corresponding region of otherretroviral gene products, and Cys³⁹² and/or Cys³⁹⁵ or both cysteines maybe replaced by serine residues.

In one specific illustrative embodiment of the invention, thesubstantial elimination of reverse transcriptase activity of the polgene product may be effected by deletion of at least a part of the polgene encoding reverse transcriptase and the at least a part of the polgene deleted may be contained between nucleotides 2586 and 4265 of thepol gene of HIV-1 isolate LAI or the corresponding region of otherretroviral pol genes.

In an additional aspect, the substantial elimination of integraseactivity of the pol gene product may be effected by deletion of at leasta part of the pol gene encoding integrase and in an illustrativeembodiment the at least a part of the pol gene deleted may be containedbetween nucleotides 4266 and 5129 of the pol gene of HIV-1 isolate LAIor the corresponding region of other retroviral pol genes.

The substantial elimination of RNase H activity of the pol gene productmay be effected by deletion of at least a part of the pol gene encodingRNase H.

In a further aspect of the invention, there is provided modifiedretroviral genomes of the invention including a segment encoding atleast one antigenic marker.

In one specific illustrative embodiment of this aspect of the invention,the sequence encoding the at least one antigenic marker is inserted intothe gag gene at an antigenically active insertion site and specificallyat the PstI site at nucleotide 1415 of the gag gene of HIV-1 LAI isolateor the corresponding location of other retroviral gag genes. Onespecific segment comprises from 1 to 4 copies of a DNA sequence selectedfrom the group consisting of:

(a) 5' GCATTCGACACTAGAAATAGAATAATAGAAGTTGAAAAT 3'; (SEQ ID NO: 5);

(b) 3' CGTAAGCTGTGATCTTTATCTTATTATCTTCAACTTTTA5'; (SEQ ID NO: 6); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequence of (a) or (b).

A variety of hybridization conditions may be employed to achieve varyingdegrees of selectivity of hybridization. For a high degree ofselectivity, stringent conditions are used to form duplexes, such as lowsalt and/or high temperature conditions, such as provided by 0.02 M to0.15 M NaC1 at temperatures of between about 50° C. to 70° C. For someapplications, less stringent hybridization conditions may be requiredsuch as 0.15 M to 0.9 M salt, at temperatures ranging from between about20° C. to 55° C. Hybridization conditions can also be rendered morestringent by the addition of increasing amounts of formamide, todestabilize the hybrid duplex.

In a yet further embodiment of the present invention, there is provideda nucleic acid molecule encoding a non-infectious retrovirus-likeparticle of the invention, comprising a modified retroviral genomedeficient in long terminal repeats and containing gag, pol and env genesin their natural genomic arrangement with the env gene being modified toprovide therein a segment encoding an antigenic anchor sequence toanchor the env gene product to the retrovirus-like particle, whereby themodified env gene encodes a modified env gene product in whichendogenous anchoring function of env has been replaced by the antigenicanchor sequence.

In one specific illustrative embodiment of this aspect of theinvention,the segment encoding the antigenic marker sequence is insertedinto the env gene, specifically between nucleotide 7777 and 7778 of theenv gene of the HIV-1 LAI isolate or the corresponding location of otherretroviral env genes. One specific segment encoding the anchor sequenceincludes a DNA sequence selected from the group consisting of:

(a) 5' TGGATCCTGTGGATTCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGTTTTGCTGGGGTTCATCATGTGG 3'; (SEQ ID NO: 7);

(b) 3' ACCTAGGACACCTAAAGGAAACGGTATAGTACGAAAAACGAAACACAACAAAACGACCCCAAGTAGTACACC 5'; (SEQ ID NO: 8); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequences of (a) or (b).

Another specific segment encoding the anchor sequence includes a DNAsequence selected from the group consisting of:

(a) 5' TCAACAGTGGCAAGTTCCCTAGCACTGGCAATCATGATAGCTGGTCTATCTTTTTGGATGTGTTCCAATGGGTCATTGCAG 3'; (SEQ ID NO: 9)

(b) 3' AGTTGTCACCGTTCAAGGGATCGTGACCGTTAGTACTATCGACCAGATAGAAAAACCTACACAAGGTTACCCAGTAACGTC 5'; and (SEQ ID NO: 10); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequences of (a) or (b). Another specific segmentencoding the anchor sequence is selected from the group consisting of:

(a) 5' TGGATCCTGTGGATTTCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGTTTGCTGGGGTTCATCATGTGGGCCTGCCAAAAAGGCAACATTAGGTGCAACATTTGCATTTGATAGTAAAGAAGAGTGGTGCAGAGAGAAAA AAGAGCAGTGGGAA 3';(SEQ ID NO: 11);

(b) 3' ACCTAGGACACCTAAAGGAAACGGTATAGTACGAAAAACGAAACACAACAAACGACCCCAAGTAGTACACCCGGACGGTTTTTCCGTTGTAATCCACGTTGTAAACGTAAACTATCATTTCTTCTCACCACGTCTCTCTTTT TTCTCGTCACCCTT 5';and (SEQ ID NO: 12); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequence of (a) or (b).

The present invention further includes, in an additional aspect, animmunogenic composition capable of eliciting a retroviral specificimmune response, comprising the retrovirus-like particles or nucleicacid molecule provided herein, and a carrier therefor. Such compositionmay be formulated for mucosal or parenteral administration, by oral,anal, vaginal or intranasal routes. The immunogenic composition maycomprise at least one other immunogenic or immunostimulating material,specifically an adjuvant, such as aluminum phosphate, aluminumhydroxide, Freund's incomplete adjuvant or QS21.

In a further aspect, the present invention includes a method ofimmunizing a host to produce a retroviral specific immune response,comprising administering to the host an immunoeffective amount of theimmunogenic composition provided herein.

The present invention also includes diagnostic procedures and kitsutilizing those materials. Specifically, in another aspect of theinvention, there is provided a method of determining the presence ofantibodies specifically reacting with retroviral antigens in a sample,comprising the steps of (a) contacting the sample with thenon-infectious retrovirus-like particle provided herein to producecomplexes comprising the non-infectious retrovirus-like particles andany said antibodies present in the sample specifically reactivetherewith; and (b) determining production of the complexes.

In an additional aspect of the invention, there is provided a method ofdetermining the presence of retroviral antigens in a sample, comprisingthe steps of (a) immunizing a host with the immunogenic compositionprovided herein to produce retroviral antigen-specific antibodies; (b)contacting the sample with the retroviral antigen-specific antibodies toproduce complexes comprising any retrovirus antigens in the sample andretroviral antigen-specific antibodies; and (c) determining productionof the complexes.

A further aspect of the invention provides a diagnostic kit fordetecting the presence of retroviral antigens in a sample comprising (a)at least one such retroviral antigen-specific antibody provided herein;(b) means for contacting the at least one antibody with the sample toproduce a complex comprising any retroviral antigens in the sample andthe retroviral antigen-specific antibodies; and (c) means fordetermining production of the complex.

Advantages of the present invention include:

an immunogenic retrovirus-like particle comprising gag, pol and env geneproducts in their natural conformations rendered non-infectious andnon-replicating by a plurality of mutations; and

an immunogenic retrovirus-like particle immunologically distinguishablefrom a virulent retrovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a construction scheme of a plasmid (pMTHIV-A) encoding aretrovirus-like particle having a modification to the gag gene productin accordance with one embodiment of the invention;

FIG. 2 shows a construction scheme of a plasmid (pMTHIVBRU) encoding aretrovirus-like particle having a modificaiton to both the gag and polgene products in accordance with a further embodiment of the invention;

FIG. 3 shows a construction scheme of a plasmid (p83-19) encoding aretrovirus-like particle having a modification in the env gne product inaccordance with a further embodiment of the invention;

FIG. 4 shows a construction scheme of plasmid pSeBS-HA2 containing aheterologous anchor sequence in the env gene;

FIG. 5 shows a flow diagram for gene assembly-aided mutagenesis;

FIG. 6 shows a construction scheme of a plasmid (pMTHIVHA2-701) encodinga retrovirus-like particle containing an antigenic marker comprising aportion of the transmembrane component of human influenza hemagglutininglycoprotein;

FIG. 7 shows a construction scheme of a plasmid (pMTHIVmHA2) encoding aretrovirus-like particle containing a non-naturally occurring marker;

FIG. 8 shows a construction scheme for a plasmid (pMTHIVMNmHA2-5)encoding a retrovirus-like particle containing a non-naturally occurringmarker;

FIG. 9 shows details of an oligonucleotide encoding an antigenic epitopefrom tobacco mosaic virus inserted into the gag gene product of anon-infectious non-replicating retrovirus-like particle;

FIG. 10 shows a construction scheme of plasmids encoding retrovirus-likeparticles having antigenic epitopes from tobacco mosaic virus;

FIG. 11 shows an immunoblot analysis of antigenically markedretrovirus-like particles (pseudovirions); and

FIG. 12 shows an immunoblot analysis of antigenically markedretrovirus-like particles to demonstrate inclusion of the antigenicmarker in the gag gene product.

GENERAL DESCRIPTION OF INVENTION

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of HIV infections, and thegeneration of immunological reagents. A further non-limiting discussionof such uses is further presented below.

Referring to FIGS. 1 and 2, there is illustrated the construction of avector pMTHIVBRU (ATCC designation 75852) containing a modifiedretroviral genome deficient in long terminal repeats, primer bindingsite and an RNA packaging sequence, and containing gag, pol and envgenes in their natural genomic arrangement. The pol gene of PMTHIVBRUhas been modified by deletion of a portion thereof to substantiallyremove the reverse transcriptase and integrase activities thereof.Furthermore, in this particular illustrated embodiment of the invention,an oligonucleotide has been inserted within the deleted pol gene tointroduce three stop codons in three different reading frames to preventremaining sequences of integrase from being translated. The gag gene ofPMTHIVBRU has also been modified to replace the two cysteine residues(Cys³⁹² and Cys³⁹⁵) in the first Cys-His box by serines.

Thus, plasmid pMTHIVBRU encodes an HIV-like particle deficient in aplurality of elements required for infectivity and/or replication of HIVbut dispensible for virus-like particle production.

Plasmid pMTHIVBRU encodes an HIV-like particle with an envelope proteincorresponding to that of the HIV-1_(LAI) isolate. Referring to FIG. 3,there is shown a plasmid p83-19 in which the LAI envelope of pMTHIVBRUhas been substantially replaced by the MN envelope sequence. Thus,plasmid p83-19 encodes an HIV-like particle deficient in a plurality ofelements required for infectivity and/or replication of HIV butdispensible for virus-like particle production, and contains as the envgene product substantially the envelope of HIV-1 isolate MN.

Referring to FIGS. 4 to 6, there is illustrated the construction of avector pMTHIVHA2-701 containing a modified HIV genome deficient in longterminal repeats, primer binding site and an RNA packaging sequence, andcontaining gag, pol and env genes in their natural genomic arrangement.The env gene in pMTHIVHA2-701 has been modified to provide therein agene encoding a different anchor sequence to anchor the env gene productto the retrovirus-like product, whereby the modified env gene encodes amodified env gene product in which endogenous anchoring function of envhas been replaced by the different anchor sequence. In retrovirus-likeparticles encoded by pMTHIVHA2-701 an immunodominant epitope ofgp41(which provides endogenous anchoring function) is no longerexpressed. Thus, such retrovirus-like particles are antigenically markedin a negative manner by the absence of an amino acid sequencecorresponding to an epitope of a retroviral protein. The differentanchor sequence may itself be antigenic to further provide a positivenon-retroviral or non-HIV retroviral antigenic marker for theretrovirus-like particles.

In this particular illustrated embodiment of the invention, a 135-bpsequence comprising a coding DNA fragment and a stop codon from thehuman influenza virus HA2 gene was inserted between nucleotides 7777 (G)and 7778 (A) of the HIV-1_(LAI) envelope gene to prevent synthesis ofthe HIV-1_(LAI) transmembrane glycoprotein. Plasmid pMTHIVHA2-701 thusencodes an HIV-like particle wherein the gp41 transmembrane glycoproteinanchoring function has been replaced by an anchor sequence from thehuman influenza virus HA2 protein and the HA2 protein further providesan antigenic marker.

Referring to FIG. 7, there is illustrated plasmid pMTHIVmHA2 which issimilar to pMTHIVHA2-701 but contains as the antigenic marker sequencereplacing the endogenous anchoring function of env, an amino acidsequence with no homology to known naturally occurring proteins.

Referring to FIG. 8, there is illustrated a vector pMTHIVMNmHA2-5 (ATCCdesignation 75853) containing a modified HIV genome deficient in longterminal repeats, primer binding site and an RNA packaging sequence andcontaining gag, pol and env genes in their natural genomic arrangement.The pol gene of pMTHIVMNmHA2-5 has been modified by deletion of aportion thereof to substantially remove the reverse transcriptase andintegrase activities thereof. Furthermore, an oligonucleotide wasinserted within the deleted pol gene to introduce three stop codons inthree different reading frames to prevent remaining sequences ofintegrase from being translated. The gag gene of pMTHIVMNmHA2-5 has alsobeen modified to replace the two cysteine residues in the first Cys-Hisbox of gag by serines. In pMTHIVMNmHA2-5, the endogenous anchoringfunction of env has been replaced by an amino acid sequence with noknown homology to naturally occurring proteins. HIV-like particlesproduced from Vero cells (and other vaccine-quality cell lives,including MCRS cells, primary monkey kidney (African Green) cells, WI38cells and baby hamster kidney cells) transfected with plasmidpMTHIVMNmHA2-5 were purified and used to immunize guinea pigs. Antiserawere collected and assayed by ELISA for anti-V3 (i.e. anti-envelope)antibodies and anti-mHA2 (i.e. anti-antigenic marker) antibodies asshown in Table 1. These results indicate that the env gene product ispresent in substantially its native conformation and that the antigenicmarker is immunogenic.

Although particular retrovirus-like particles have been described inwhich endogenous anchoring function of env has been replaced by theantigenic anchor sequence of particular natural and unnatural proteins,it is appreciated that many variations, adaptations and modificationscan be made to the particular means by which the endogenous anchoringfunction can be replaced without departing from the essence of theinvention.

Referring to FIGS. 9 and 10, there is illustrated plasmids (pHIV-T1;pHIV-T2; pHIV-T3 and pHIV-T4) containing between one and four copies ofa DNA sequence encoding an antigenic epitope from TMV. In the particularembodiments shown, the TMV epitope is inserted into the gag gene of HIVto produce a hybrid gag gene product, and the plasmids are deficient inthe plurality of elements required for infectivity and/or replication ofHIV but dispensible for virus-like particle production as describedabove. Stable cell lines were produced using plasmids pHIV-T1, pHIV-T2(ATCC designation 75852), pHIV-T3 and pHIV-T4 (containing 1, 2, 3 and 4copies of the antigen epitope, respectively) that produced HIV-likeparticles containing the antigenic marker inserted into the gag protein.These HIV-like particles were purified and their reactivity withanti-HIV monoclonal antibodies (FIG. 11) and anti-TMV marker antiserum(FIG. 12) determined. The results are shown in FIGS. 11 and 12 andindicate that the HIV-like particles contain gp120, gp41 and p24 insubstantially their natural conformations and that the TMV marker isable to be recognized by anti-marker antibodies.

While specific embodiments of the marker sequences, which may also be ananchor sequence, are described herein, it is apparent that any otherconvenient amino acid sequence providing marker and/or anchoringfunction may be employed herein, including the absence of an amino acidsequence that corresponds to an epitope of a retroviral protein. Theamino acid sequence providing marker function may comprise anon-naturally occurring antigenic sequence which has no homology toknown proteins. An example of such sequence is the mutant HA2 sequencedescribed above. Other examples may include antigenic regions ofnon-human or non-mammalian protein, such as non-human or non-mammalianpathogenic or comensual organisms. An example of such sequence is theTMV described above.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of HIV infections, and thegeneration of immunological reagents. A further non-limiting discussionof such uses is further presented below.

Vaccine Preparation and Use

It has been shown that an immunogenic preparation in accordance with theinvention can elicit an immune response. One possible use of the presentinvention is, therefore, as the basis of a potential vaccine againstretroviral diseases including AIDS and AIDS-related conditions. In afurther aspect, the invention thus provides a vaccine against AIDS andAIDS-related conditions, comprising an immunogenic composition inaccordance with the invention.

Immunogenic compositions, suitable to be used as vaccines, may beprepared from non-infectious retrovirus-like particles as disclosedherein. The immunogenic composition elicits an immune response whichproduces antibodies that are antiviral. Should the vaccinated subject bechallenged by a retrovirus, such as HIV, the antibodies bind to thevirus and thereby inactivate it.

Vaccines may be prepared as injectables, as liquid solutions oremulsions. The non-infectious retrovirus-like particles may be mixedwith pharmaceutically-acceptable excipients which are compatible withthe retrovirus-like particles. Excipients may include water, saline,dextrose, glycerol, ethanol, and combinations thereof. The vaccine mayfurther contain auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessof the vaccines. Methods of achieving an adjuvant effect for the vaccineinclude the use of agents, such as aluminum hydroxide or phosphate(alum), commonly used as 0.05 to 0.1 percent solution in phosphatebuffered saline and other adjuvants, including QS21 and incompleteFreunds adjuvant. Vaccines may be administered parenterally, byinjection subcutaneously or intramuscularly. Alternatively, theimmunogenic compositions formed according to the present invention, maybe formulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Oral formulations may include normally employedincipients, such as pharmaceutical grades of saccharine, cellulose andmagnesium carbonate. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained-release formulations orpowders and contain 10 to 95% of the retrovirus-like particles of theinvention.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of micrograms of the retrovirus-likeparticles. Suitable regimes for initial administration and booster dosesare also variable, but may include an initial administration followed bysubsequent administrations. One example of an immunization schedule isat least one pre-immunization with a retrovirus-like particle, accordingto the present invention followed by at least one secondary immunizationwith a synthetic peptide described in published European PatentPublication Number 0 570 980, assigned to the assignee hereof. Thedosage of the vaccine may also depend on the route of administration andwill also vary according to the size of the host.

Nucleic acid molecules encoding the retrovirus-like particles of thepresent invention may also be used directly for immunization byadministration of the nucleic acid molecules directly, for example byinjection to a host. Processes for the direct injection of DNA into testsubjects for genetic immunization are described in, for example, Ulmeret al, 1993 (a list of references appears at the end of the disclosureand each of the listed references is incorporated by reference withoutfurther reference thereto).

Molecules in accordance with the invention may further find use in thetreatment (prophylactic or curative) of AIDS and related conditions, byacting either to displace the binding of the HIV virus to human oranimal cells or by disturbing the 3-dimensional organization of thevirus.

A further aspect of the invention thus provides a method for theprophylaxis or treatment of AIDS or related conditions, comprisingadministering an effective amount of an immunogenic composition inaccordance with the invention.

Immunoassays

The retrovirus-like particles of the present invention are useful asimmunogens, as antigens in immunoassays including enzyme-linkedimmunosorbent assays (ELISA), RIAs and other non-enzyme linked antibodybinding assays, or procedures known in the art for the detection ofanti-retroviral (for example, HIV) HIV antibodies and retroviral antigen(for example, HIV). In ELISA assays, the retrovirus-like particles areimmobilized onto a selected surface, for example a surface capable ofbinding proteins, such as the wells of a polystyrene microtitre plate.After washing to remove incompletely adsorbed retrovirus-like particles,a non-specific protein, such as a solution of bovine serum albumin (BSA)or casein, that is known to be antigenically neutral with regard to thetest sample may be bound to the selected surface. This allows forblocking of non-specific adsorption sites on the immobilizing surfaceand thus decreases the background caused by non-specific bindings ofantisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials to be tested, in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from about 2 to 4 hours, at temperaturessuch as of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound retrovirus-like particles, and subsequent washing, theoccurrence, and even amount, of immunocomplex formation may bedetermined by subjecting the immunocomplex to a second antibody havingspecificity for the first antibody. If the test sample is of humanorigin, the second antibody is an antibody having specificity for humanimmunoglobulins and in general IgG. To provide detecting means, thesecond antibody may have an associated activity, such as an enzymaticactivity that will generate, for example, a colour development uponincubating with an appropriate chromogenic substrate. Quantification maythen be achieved by measuring the degree of colour generation using, forexample, a visible spectra spectrophotometer.

In one diagnostic embodiment where it is desirable to identifyantibodies that recognize a plurality of HIV isolates, a plurality ofimmunologically distinct retrovirus-like particles of the presentinvention are immobilized onto the selected surface. Alternatively, whenthe anti-HIV antibodies recognize epitopes that are highly conservedamong various HIV isolates (for example, a B-cell epitope from gag orgp41) a single or a limited number of retrovirus-like particles may beimmobilized. In a further diagnostic embodiment where it is desirable tospecifically identify antibodies that recognize a single HIV isolate(for example, LAI, MN, SF2 or HXB2) a single particular retrovirus-likeparticle of the present invention may be immobilized. This furtherdiagnostic embodiment has particular utility in the fields of medicine,clinical trials, law and forensic science where it may be critical todetermine the particular HIV isolate that was responsible for thegeneration of an immune response including an antibody response.

In a further diagnostic embodiment, it may be desirable to specificallyidentify immunologically distinct retroviruses, for example, HIVisolates that belong to different clades. Immunologically distinct HIVisolates may include for example, LAI, MN, SF2, HXB2 or a primary HIV-1isolate. In this diagnostic embodiment, a particular retrovirus-likeparticle of the present invention is useful for generating antibodiesincluding monoclonal antibodies that specifically recognize such animmunologically distinct HIV isolate.

It is understood that a mixture of immunologically distinctretrovirus-like particles may be used either as an immunogen in, forexample, a vaccine or as a diagnostic agent. There may be circumstanceswhere a mixture of retrovirus-like particles are used to providecross-isolate protection and/or diagnosis. In this instance, the mixtureof immunogens is commonly referred to as a "cocktail" preparation.

The present invention advantageously provides retrovirus-like particlescomprising gag, pol and env gene products substantially in their naturalconformations. Such retrovirus particles will thus be recognized byconformational anti-HIV antibodies (such as anti-env antibodies) thatmay not recognize the HIV antigen in a denatured form or a syntheticpeptide corresponding to such an HIV antigen. The retrovirus-likeparticles of the invention are therefore particularly useful as antigensand as immunogens in the generation of anti-retroviral antibodies(including monoclonal antibodies) in diagnostic embodiments.

In addition, the presence of the marker generates a specific immuneresponse thereto the detection of which by the methods described aboveenables the ready distinction between immunization of a host with theimmunogenic compositions provided herein compared to material infectionby a virulent retrovirus. The ability to effect such diagnosis anddifferentiation has advantageous utility in the fields of epidemiology,clinical trials, forensic science and immunology.

Other Uses

Molecules which bind to the retrovirus-like particles on which theinvention is based, particularly antibodies, antibody-related moleculesand structural analogs thereof, are also of possible use as agents inthe treatment and diagnosis of AIDS and related conditions.

Variants of antibodies (including variants of antigen binding site),such as chimeric antibodies, humanized antibodies, veneered antibodies,and engineered antibodies that are specific for the retrovirus-likeparticles of the invention are included within the scope of theinvention.

Antibodies and other molecules which bind to the retrovirus-likeparticles of the present invention can be used for therapeutic(prophylactic and curative) and diagnostic purposes in a number ofdifferent ways, including the following:

For passive immunization by suitable administration of antibodies,possibly humanized antibodies, to HIV infected patients.

To activate, complement or mediate antibody dependent cellularcytotoxicity (ADCC) by use of antibodies of suitable subclass or isotype(possibly obtained by appropriate antibody engineering) to be capable ofperforming the desired function.

For targeted delivery of toxins or other agents, for example, by use ofimmunotoxins comprising conjugates of antibody and a cytotoxic moiety,for binding directly or indirectly to cell-surface exposed HIV proteinsof HIV-infected cells (for example, gp120).

For targeted delivery of highly immunogenic materials to the surface ofHIV-infected cells, leading to possible ablation of such cells by eitherthe humoral or cellular immune system of the host.

For detection of HIV, using a variety of immunoassay techniques.

Thus, in yet a further diagnostic embodiment, the immunogeniccompositions of the present invention (individually, or as mixturesincluding cocktail preparations) are useful for the generation of HIVantigen specific antibodies (including monoclonal antibodies) that canbe used to detect HIV or antigens, or neutralize HIV in samplesincluding biological samples.

In an alternative diagnostic embodiment, the retrovirus-like particlesof the present invention can be used to specifically stimulate HIVspecific T-cells in biological samples from, for example, HIV-infectedindividuals for diagnosis or therapy.

Biological Deposits

Certain plasmids that encode retrovirus-like particles according toaspects of the present invention that are described and referred toherein have been deposited with the American Type Culture Collection(ATCC) located at Rockville, Md. USA pursuant to the Budapest Treaty andprior to the filing of this application. Samples of the depositedplasmids will become available to the public upon grant of a patentbased upon this United States patent application. The inventiondescribed and claimed herein is not to be limited in scope by plasmidsdeposited, since the deposited embodiment is intended only as anillustration of the invention. Any equivalent or similar plasmids thatencode similar or equivalent retrovirus-like particles as described inthis application are within the scope of the invention.

    ______________________________________                                        Deposit Summary                                                               Plasmid        ATCC Designation                                                                           Date Deposited                                    ______________________________________                                        pMTHIVBRU      75,852       August 4, 1994                                    pMTHIVMNmHA2-5 75,853       August 4, 1994                                    pHIV-T2        75,851       August 4, 1994                                    ______________________________________                                    

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitations.Immunological and recombinant DNA methods may not be explicitlydescribed in this disclosure but are well within the scope of thoseskilled in the art.

EXAMPLES

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these EXAMPLES areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example describes the construction of plasmid pMTHIVBRU.

Plasmid pMTHIVBRU was constructed as shown in FIGS. 1 and 2. Thisplasmid is a modification of the expression vector pMTHIVd25 describedin Rovinski et al 1992 (the literature references are identified at theend of the specification) and which contains an RNA packaging deletion,and was engineered to contain a series of mutations/deletions. Thus, aCys-His box mutation included replacements of two cysteine codons (inSEQ ID NO: 13) with two serine codons in the first Cys-His box (SEQ IDNO: 14) of the gag protein as shown in FIG. 1. This was accomplished bya PCR-based mutagenesis method. Two primers were synthesized: theupstream primer having the sequence 5'-GGACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAA TAATCCACCTATCCCAGTAGGAG-3'(SEQ ID NO: 15), comprising nucleotides 1,507 to 1,567 of HIV-1_(LAI)(all nucleotide numbering is according to Wain-Hobson et al., 1985) witha SpeI site at the 5'-end; and the downstream primer having the sequence5' CTCGGGCCCTGCAATTTCTGGCTATGTGCCCTTC TTTGCCACTATTGAAACTCTTAACAATC-3'(SEQ ID NO: 16), being the reverse complement of nucleotides 2,011 to1,953 with an ApaI site at the 5'-end. In the downstream primer, twoadenosine residues representing the reverse complement of nucleotides1,963 and 1,972 (Wain Hobson et al, 1985; Myers et al, 1990) werechanged to thymidine, resulting in the replacement of the two cysteinesat amino acid positions 392 and 395 of the gag gene product with twoserines (FIG. 1). These two primers were used to amplify the SpeI-ApaIDNA fragment (nucleotides 1507 to 2006) of PMTHIV (Rovinski et al, 1992)which was used as a template. The PCR-amplified SpeI-ApaI fragment waspurified by agarose gel electrophoresis and digested with restrictionenzymes SpeI and ApaI. This fragment was used to replace thecorresponding fragment in pMTHIVd25 (Rovinski et al, 1992). Theresulting plasmid was named pMTHIV-A, which contains both the RNApackaging sequence deletion and the Cys-His box mutation.

In order to delete the reverse transcriptase and integrase, two BalIrecognition sites at nucleotides 2,655 and 4,587 of HIV-1_(LAI), wereused (FIG. 2). The 1.9-kbp fragment between the two BalI sites containsDNA sequences encoding more than 95% k of the reverse transcriptase andthe first 108 amino acids of the integrase. The plasmid pMTHIV-A wasdigested with BalI. After removing the 1.9-kbp BalI fragment by gelelectrophoresis, the remaining portion of the plasmid was ligated with adouble-stranded oligonucleotide: 5'-GTATAAGTGAGTAGCGGCCGCAC-3' (only onestrand is shown--SEQ ID NO: 17) which contains three stop codons inthree different reading frames to prevent the remaining sequences ofintegrase from being translated. The resulting plasmid was termedpMTHIVBRU.

Example 2

This Example describes the construction of plasmids encoding HIV-likeparticles containing antigenically marked envelope anchors.

Plasmid p83-19 was constructed from expression vector pMTHIVBRU, asshown in FIG. 3. This plasmid contains a hybrid envelope gene which wasengineered by replacing DNA encoding most of gp120_(LAI), with thecognate DNA encoding gp120_(MN). This was accomplished by replacing aKPnI/BglII DNA fragment (nucleotides 6379 to 7668) from HIV-1_(LAI),with a KpnI/BglII DNA fragment (nucleotides 6358 to 7641) fromHIV-1_(MN).

Plasmid pMTHIVHA2-701 was constructed from expression vectors pBT1(Alizon et al, 1984) and pMTHIVd25 (Rovinski et al, 1992), as shown inFIGS. 4 to 6. The pMTHIVHA2-701 vector contains a 135-bp sequencecomprising a coding DNA fragment and a stop codon from the humaninfluenza virus HA2 gene (Min Jou et al, 1980), inserted betweennucleotides 7777(G) and 7778(A) of the HIV-1_(LAI) envelope gene(Wain-Hobson et al, 1985; Myers et al, 1990). The stop codon wasinserted to prevent synthesis of the HIV-1_(LAI) gp41 transmembraneglycoprotein. A SalI (nucleotide 5821)/BamHI (nucleotide 8522) DNAfragment from pBT1 was subcloned into pSelect (Promega) to produce pSeBS(FIG. 4). The latter plasmid was used for insertion of the 135-bp by aprocedure termed herein as `gene assembly-aided mutagenesis (GAAM)`. Amutagenic primer, which was designed to contain the 135-bp sequencecomprising a coding DNA fragment from the human influenza virus HA2 gene(Min Jou et al, 1980), was assembled as shown in FIG. 5. OligonucleotideI is a 99 mer containing (from 3' to 5') 30 bases complementary tonucleotides 7748 to 7777 of HIV-1_(LAI) (Wain-Hobson et al, 1985; Myerset al, 1990) and 69 bases which are complementary to HA2 gene sequences(Min Jou et al, 1980) encoding amino acids 180 to 202 of the HA2protein. Oligonucleotide II is a 96 mer comprising (from 3' to 5') i) 60bases complementary to HA2 gene sequences which encode amino acids 203to 221 of the HA2 protein and contain the HA2 stop codon (Min Jou et al,1980), ii) 6 bases (ATCATT--SEQ ID NO: 18) defining two more stopcodons, and iii) 30 bases complementary to nucleotides 7778 to 7807 ofHIV-1_(LAI) , (Wain-Hobson et al, 1985; Myers et al, 1990).Oligonucleotide III is a bridging 30 mer having 15 nucleotidescomplementary to the 5'-end of oligonucleotide I and 15 nucleotidescomplementary to the 3'-end of oligonucleotide II. Ten picomoles ofoligonucleotides I and II were mixed with 20 picomoles ofoligonucleotide III and phosphorylated at 37° C. for 1.5 h in 20 μlkinase buffer (50 mM Tris-HCl, pH 7.5, 10 MM MgCl₂, 10 mm KCl, 5 MM DTT,and 0.5 MM ATP) containing 2 units of T4 polynucleotide kinase. Theoligonucleotides were annealed by heating the mixture to 95° C. for 5min and subsequently cooling it slowly to room temperature. To thismixture was added 3 μl of 10×ligase buffer (0.5 M Tris-HCl, pH 7.4, 0-1M MgCl₂, 0.1 M DTT, 10 mM Spermidine, and 1 mg/ml BSA), 3 μl of 10 mMATP, and 5 units of T4 DNA ligase, and the ligation mixture wasincubated overnight at 16° C. to complete the assembly of the mutagenicprimer (FIG. 5). This primer was used in the mutagenesis procedurewithout further purification.

Mutagenesis was performed using the Altered Sites in vitro MutagenesisSystem from Promega (Madison, Wis.). The template for mutagenesisconsisted of the pSeBS plasmid (FIG. 4) which contained the 2.7-kbpSalI/BamHI DNA fragment of the HIV-1_(LAI) envelope gene (nucleotides5821 to 8522) cloned into the pSelect phagemid vector provided in themutagenesis kit. Following the mutagenesis procedure, putative cloneswere identified by colony hybridization with a ³² P-labelledoligonucleotide III probe. Positive clones were confirmed by DNAsequencing. One of these clones, designated pSeBS-HA2, was used for theconstruction of the final vector. To this end, the modified SalI/BamHIinsert from pSeBS-HA2 was subcloned into pMTHIVd25-dSalI; the latter isa plasmid derived from pMTHIVd25 (Rovinski et al, 1992) by partialdigestion with SalI followed by Klenow treatment to eliminate the SalIsite within the plasmid backbone. The final expression construct wasdesignated pMTHIVHA2-701.

An expression vector, pMTHIVmHA2 (shown in FIG. 7) containing aheterologous DNA sequence inserted between nucleotides 7777 (G) and 7778(A) of the HIV-1_(LAI) envelope gene (Rovinski et al, 1992; Wain-Hobsonet al, 1985) was engineered as described above. In this case, a 134-bpsequence, comprising a coding DNA fragment from the human influenzavirus HA2 gene (Min Jou et al, 1990) and 68 nucleotides that, when fusedto the HA2 sequences, encodes an amino acid sequence with no homology toknown naturally occurring proteins, was inserted downstream ofnucleotide 7777 of HIV-1_(LAI) (FIG. 7). The insertion resulted in aframeshift in the translation of HIV-1_(LAI) coding sequences, and thecreation of a stop codon (TAG) to prevent synthesis of the gp41transmembrane glycoprotein of HIV-1_(LAI) . The final expressionconstruct was designated pMTHIVmHA2 (FIG. 7).

Plasmid pMTHIVMNmHA2-5 was constructed from expression vectors p83-19and pMTHIVmHA2 as shown in FIG. 8. This plasmid was designed to have allof the mutations of elements required for infectivity and/or replicationof p83-19 and to contain the 134-bp insert sequence of pMTHIVmHA2 (FIG.7). To this end, p83-19 was digested with BglII (nucleotide 7,641) andXhoI (nucleotide 8,944) to remove a 1276-bp DNA fragment which wasreplaced by the cognate BqlII/XhoI fragment of pMTHIVmHA2.

Example 3

This Example describes the construction of plasmids encoding HIV-likeparticles containing antigenic epitopes from TMV.

Plasmids pHIV-Tl, pHIV-T2, pHIV-T3, and pHIV-T4 represent modifiedversions of the p83-19 construct in that they contain, respectively,either one, two, three, or four copies of a double-strandedoligonucleotide (FIGS. 9, 10 and 11) comprising at least one antigenicepitope (Westhof et al, 1984; Trifilleff et al, 1991) from TMV coatprotein. The construction of these four vectors is illustrated in FIGS.9 and 10. To engineer all constructs, plasmid PMTHIV-A (FIG. 1) wasfirst digested with SacII and ApaI to isolate a 1,328-bp DNA fragmentwhich was then subcloned into pBluescript (Stratagene). The recombinantplasmid was then digested with PstI which cleaves HIV-1_(LAI) DNA atnucleotide 1,415 within the gag gene. Subsequently, either one, two,three, or four copies of the double-stranded oligonucleotide shown inFIG. 9 (coding strand: SEQ ID NO: 19, complementary strand: SEQ ID NO:20, encoded amino acids: SEQ ID NO: 21) were inserted into thisrestriction site. Finally, the resulting recombinant plasmids weredigested with SacII and ApaI to release the modified insert which wasthen cloned into the cognate region of plasmid p83-19 (FIG. 10).

The expression of retrovirus-like particles containing either the mHA2epitope or various copies of the TMV epitope is depicted in FIG. 11.Vero cells were grown to 80% confluency and transfected with 20 μg ofplasmid DNA by the transfinity (BRL) calcium phosphate procedure.Culture supernatants were analyzed for protein expression at 48 hpost-transfection. Culture media (10 ml) from cells transfected withindividual expression constructs were collected and clarified bycentrifugation at 2,000×g (sorvall RT 6000B; Dupont Company, Wilmington,Del.) for 15 min at 4° C. Retrovirus-like particles were isolated byultra-centrifugation. Pelleted particles were suspended to 40 μl of TNE,mixed with 10 μl of 5×Laemmli sample buffer and boiled for 3 min. Viralproteins were then separated by SDS PAGE and transferred to Immobilonmembranes (Millipore, Bedford, Mass.). Membranes were blocked withBLOTTO buffer (PBS containing 5% Carnation instant nonfat dry milk,0.0001% wt/vol thimerosal, and 0.01% vol/vol antifoam A emulsion) for 2h at 25° C. and then incubated with appropriate dilutions of antibodiesovernight at 4° C. Filters were then incubated with a goat anti-mouseimmunoglobulin G antibody conjugated to alkaline phosphatase (Promega,Madison, Wis.) and reacted with the alkaline phosphatase chromogenicsubstrates nitroblue tetrazolium chloride and5-bromo-4-chloro-3-indolyphosphate ρ-toluidine salt (BRL). A cocktail ofanti-gp120, anti-gp41, and anti-p24 antibodies was used in Panel A. Amixture of anti-gp120 and anti-p24 antibodies was used in Panel B.

The results shown in FIG. 11 demonstrate that the antigenically markedHIV-like particles produce gp120, gp41 and p24 substantially in theirnatural conformations.

Example 4

This Example describes the immunogenicity and immunoreactivity ofantigenically marked HIV-like particles.

One of plasmids pHIV-T1, pHIV-T2, pHIV-T3, or PHIV-T4 (FIG. 10) wasco-transfected with plasmid pSV2neo into Vero cells, and stable celllines were established that produce HIV-like particles. HIV-likeparticles were purified, and their reactivity to immune sera from guineapigs immunized with a peptide corresponding to the TMV marker insertedinto the gac gene product was determined by immuno blot analysis. Toobtain the immune sera, guinea pigs were immunized with 100 μg of apeptide consisting of the TMV marker conjugated to KLH and adjuvanted inFreund's complete adjuvant. All animals were boosted three times at3-week intervals with the same peptide adjuvanted in Freund's incompleteadjuvant. Immune sera were collected two weeks after the last boostershots. The results, presented in FIG. 12, illustrate the reactivity ofthe immune sera to various forms of the gag gene product present in thevarious HIV-like particles and demonstrate the antigenicity of the TMVmarker in the context of a modified HIV-1-like particle.

Plasmid pMTHIVMNmHA2-5 was co-transfected with plasmid pSV2neo into Verocells, and a stable cell line was established that produces HIV-likeparticles. HIV-like particles were then purified, and guinea pigsimmunized with 10 μg of gag p24-equivalent amounts of HIV-like particlesadjuvanted in Freund's complete adjuvant. All animals were boosted threetimes at 3-week intervals with HIV-like particles adjuvanted in Freund'sincomplete adjuvant. Two weeks after the last booster shots, immune serawere collected and assayed by ELISA for anti-V3 and anti-mHA2 markerreactivities. The results, presented in Table 1 below, indicate thatguinea pigs immunized with HIV-like particles containing the mHA2 markerproduced antibodies capable of recognizing peptides representing themHA2 marker (MHA-1) and V3 loop neutralization domains (CLTB56, CLTB71,and CLTB73). These data, therefore, demonstrate that the mHA2 marker isimmunogenic when presented in the context of an HIV-like particle andthat antibodies are also produced against the major neutralizingdeterminants of the V3 loops from different HIV isolates.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides certainnon-infectious, non-replicating, retrovirus-like particles and nucleicacid molecules encoding them as, for example, immunogenic preparationsuseful for vaccination, the generation of retroviral-specific antiseraand as antigens in diagnostic methods and kits. The retrovirus-likeparticles may have been rendered non-infectious by modifications to thepol and/or gag gene products. Particular retrovirus-like particlescontain non-retroviral antigenic markers. Modifications are possiblewithin the scope of this invention.

                                      TABLE 1                                     __________________________________________________________________________    The ability ot retrovirus-like particles containing an antigenic marker       to generate a                                                                 retroviral-specific immnune response and a marker-specific immune             response.                                                                                                                ELISA IgG TITRES.sup.1             PEPTIDE                                                                            SEQUENCE                 SPECIFICITY                                                                          SEQ ID NO.                                                                          GP542                                                                             GPS43                                                                             GP544                      __________________________________________________________________________    MHA-1                                                                              GPAKKATLGATFAFDSKEEWCREKKEQWE                                                                          mHA2 marker                                                                          22    500 5,000                                                                             2,500                      CLTB56                                                                             NKRKRIHIGPGRAFYTTKN      V3 (MN)                                                                              23    500   500                                                                             2,500                      CLTB71                                                                             NTRKSIYIGPGRAFHTTGR      V3 (SF2)                                                                             24    500 2,500                                                                             2,500                      CLTB73                                                                             NTRKRIRIQRGPGRAFVTIGK    V3 (HXB2)                                                                            25    500 1,000                                                                             2,500                      Irrelevant                                                                         MKKTRFVLNSIALGLSVLSTSFVAQATLPSFVSEQNS                                                                  Non-HIV                                                                              26    100   100                                                                               100                      __________________________________________________________________________     .sup.1 Each guinea pig (GP542, GP543 and GP544) was immunized as describe     in Example 4.                                                            

REFERENCES

1. Rovinski, B., Haynes, J. R., Cao, S. X., James, O., Sia, C.,Zolla-Pazner, S., Matthews, T. J. and Klein, M. (1992) J. Virol., 66,4003-4012.

2. Wain-Hobson, S., Sonigo, P., Danos, O., Col, S. and Alizon, M. (1985)Cell, 40, 9-17.

3. Myers, G., Berzofsky, J. A., Rabson, A. B., Smith, T. F. andWong-Staal, F. (ed.) (1990) Human retroviruses and AIDS. TheoreticalBiology and Biophysics, Group T-10. Los Alamos National Laboratory, LosAlamos, N.M.

4. Alizon, M., Sonigo, P., Barre-Sinoussi, F., Chermann, J. C.,Tiollais, P., Montagnier, L. and Wain-Hobson, S. (1984) Nature, 312,757-780.

5. Min Jou, W., Verhoeyen, M., Devos, R., Saman, E., Fang, R.,Huylebroeck, D. and Fiers, W. (1980) Cell, 19, 683-696.

6. Westhof, E., Altschuh, D., Moras, D., Bloomer, A. C., Mondragon, A.,Klug, A. and Van Regenmortel, M. H. (1984) Nature, 311, 123-126.

7. Trifilleff, E., Dubs, M.C. and Regenmertel, M. H. V. (1991) Mol.Immunol., 28, 889-896.

8. Kieny, M. P. (1990) J. Acquired Immune Deficiency Syndromes, 3,395-402.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 26                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                            - Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu Va - #l Glu Asn                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cy - #s Phe Leu Leu Cys Val         #                15                                                           - Val Leu Leu Gly Phe Ile Met Trp                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 27 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Il - #e Met Ile Ala Gly Leu         #                15                                                           - Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gl - #n                             #            25                                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 52 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cy - #s Phe Leu Leu Cys Val         #                15                                                           - Val Cys Trp Gly Ser Ser Cys Gly Pro Ala Ly - #s Lys Ala Thr Leu Gly         #            30                                                               - Ala Thr Phe Ala Phe Asp Ser Lys Glu Glu Tr - #p Cys Arg Glu Lys Lys         #        45                                                                   - Glu Gln Trp Glu                                                                 50                                                                        - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #    39            ATAG AATAATAGAA GTTGAAAAT                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #    39            TATC TTATTATCTT CAACTTTTA                                  - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 71 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - TGGATCCTGT GGATTCCTTT GCCATATCAT GCTTTTTGCT TTGTGTTGTT TT - #GCTGGGGT         60                                                                          #       71                                                                    - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 72 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - ACCTAGGACA CCTAAAGGAA ACGGTATAGT ACGAAAAACG AAACACAACA AA - #ACGACCCC         60                                                                          #       72                                                                    - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 81 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - TCAACAGTGG CAAGTTCCCT AGCACTGGCA ATCATGATAG CTGGTCTATC TT - #TTTGGATG         60                                                                          #81                TGCA G                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 81 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - AGTTGTCACC GTTCAAGGGA TCGTGACCGT TAGTACTATC GACCAGATAG AA - #AAACCTAC         60                                                                          #81                ACGT C                                                     - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 156 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - TGGATCCTGT GGATTTCCTT TGCCATATCA TGCTTTTTGC TTTGTGTTGT TT - #GCTGGGGT         60                                                                          - TCATCATGTG GGCCTGCCAA AAAGGCAACA TTAGGTGCAA CATTTGCATT TG - #ATAGTAAA        120                                                                          #      156         AGAA AAAAGAGCAG TGGGAA                                     - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 156 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - ACCTAGGACA CCTAAAGGAA ACGGTATAGT ACGAAAAACG AAACACAACA AA - #CGACCCCA         60                                                                          - AGTAGTACAC CCGGACGGTT TTTCCGTTGT AATCCACGTT GTAAACGTAA AC - #TATCATTT        120                                                                          #      156         TCTT TTTTCTCGTC ACCCTT                                     - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 63 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - GGACTAGTAC CCTTCAGGAA CAAATAGGAT GGATGACAAA TAATCCACCT AT - #CCCAGTAG         60                                                                          #             63                                                              - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 62 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - CTCGGGCCCT GCAATTTCTG GCTATGTGCC CTTCTTTGCC ACTATTGAAA CT - #CTTAACAA         60                                                                          #              62                                                             - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #                23GCCG CAC                                                   - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 6 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #            6                                                                - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #                48GAAA TAGAATAATA GAAGTTGAAA ATGGTGCA                        - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #                48TGAT CTTTATCTTA TTATCTTCAA CTTTTACC                        - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Gl - #u Val Glu Asn Gly Ala         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Gly Pro Ala Lys Lys Ala Thr Leu Gly Ala Th - #r Phe Ala Phe Asp Ser         #                15                                                           - Lys Glu Glu Trp Cys Arg Glu Lys Lys Glu Gl - #n Trp Glu                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gl - #y Arg Ala Phe Tyr Thr         #                15                                                           - Thr Lys Asn                                                                 - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Asn Thr Arg Lys Ser Ile Tyr Ile Gly Pro Gl - #y Arg Ala Phe His Thr         #                15                                                           - Thr Gly Arg                                                                 - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Asn Thr Arg Lys Arg Ile Arg Ile Gln Arg Gl - #y Pro Gly Arg Ala Phe         #                15                                                           - Val Thr Ile Gly Lys                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 37 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Met Lys Lys Thr Arg Phe Val Leu Asn Ser Il - #e Ala Leu Gly Leu Ser         #                15                                                           - Val Leu Ser Thr Ser Phe Val Ala Gln Ala Th - #r Leu Pro Ser Phe Val         #            30                                                               - Ser Glu Gln Asn Ser                                                                 35                                                                    __________________________________________________________________________

What we claim is:
 1. A nucleic acid molecule, comprising:a modifiedhuman immunodeficiency virus (HIV) genome, deficient in long terminalrepeats (LTRs), containing the gag, pol and env genes in their naturalgenomic arrangement and means for expression operatively connected tosaid modified HIV genome for production of gene products in cells toproduce non-infectious, immunogenic, non-replicating HIV retrovirus-likeparticles comprising, in an assembly, the gag, pot and env geneproducts, wherein: (1) at least one codon in the gag gene has beenmutated to effect modification to the gag gene product in the firstCys-His box only of the gag gene product by replacing at least one aminoacid residue in the first Cys-His box contributing to gag-dependentgenomic viral RNA packaging into said HIV retrovirus-like particles toeffect reduction of gag-dependent genomic viral RNA packaging into theparticles while retaining the immunogenicity of the HIV retrovirus-likeparticles; (2) codons in the pol gene encoding a portion of the pol geneproduct contributing to reverse transcriptase activity have been deletedto substantially eliminate reverse transcriptase activity of the polgene product in the HIV retrovirus-like particles; (3) codons in the polgene encoding a portion of the pol gene product contributing tointegrase activity have been deleted to substantially eliminateintegrase activity of the pol gene product in the HIV retrovirus-likeparticles; and (4) codons in the pol gene encoding a portion of the polgene product contributing to RNase H activity have been deleted tosubstantially eliminate RNase H activity of the pol gene product in theHIV retrovirus-like particles.
 2. The nucleic acid molecule of claim 1wherein said nucleic acid molecule comprises a Sacl (678) to Xhol (8944)HIV-1_(LAI) restriction fragment.
 3. The nucleic acid molecule of claim1, wherein said molecule is deficient in the primer binding site (PBS)and/or an RNA packaging signal.
 4. The nucleic acid molecule of claim 1,wherein the reduction of gag-dependent RNA packaging is effected bymutagenesis of a region thereof encoding at least one amino acidcontained within a region of the gag gene product corresponding toCys³⁹² to Cys³⁹⁵ of the HIV-1 LAI isolate, or a corresponding region ofother HIV-1 isolates.
 5. The nucleic acid molecule of claim 4, wherein acodon encoding Cys³⁹² and/or Cys³⁹⁵ is replaced by a codon encodingserine.
 6. The nucleic acid molecule of claim 5, wherein codons encodingboth Cys³⁹² and Cys³⁹⁵ are replaced by codons encoding serine.
 7. Thenucleic acid molecule of claim 1, wherein the HIV retrovirus is selectedfrom the group consisting of HIV-1 and HIV-2.
 8. The nucleic acidmolecule of claim 1, wherein the env gene is an LAI env gene, an MN envgene or an env gene from a primary HIV-1 isolate.
 9. An immunogeniccomposition capable of eliciting a retroviral specific immune response,comprising the nucleic acid molecule of claim 1 and a carrier therefor.10. The immunogenic composition of claim 9 formulated for mucosal orparenteral administration.
 11. The immunogenic composition of claim 9formulated for oral, anal, vaginal, or intranasal administration. 12.The immunogenic composition of claim 9 further comprising at least oneother immunogenic and/or immunostimulating material.
 13. The immunogeniccomposition of claim 9, further comprising an adjuvant.
 14. Thecomposition of claim 13, wherein the adjuvant is aluminum phosphate,aluminum hydroxide, Freund's incomplete adjuvant, or QS21.
 15. A methodof immunizing a host to produce a retroviral specific immune response,comprising administering to the host an immunoeffective amount of theimmunogenic composition of claim
 9. 16. The nucleic acid molecule ofclaim 1, wherein the at least a part of the pol gene encoding reversetranscriptase is contained between nucleotides 2586 and 4265 of the polgene of HIV-1 isolate LAI or the corresponding region of other HIV polgenes.
 17. The nucleic acid molecule of claim 1, wherein the at least apart of the pol gene encoding integrase is contained between nucleotides4266 and 5129 of the pol gene of HIV-1 isolate LAI or the correspondingregion of other HIV pol genes.
 18. The nucleic acid molecule of claim 1,wherein said modified retroviral genome includes a heterologous nucleicacid insert encoding at least one non-retroviral, non-mammalianantigenic marker, wherein said marker, when presented in the context ofsaid HIV retrovirus-like particle, is capable of generating an immuneresponse specific to said antigenic marker when the particle isadministered to a host.
 19. The nucleic acid molecule of claim 18,wherein the heterologous nucleic acid insert is inserted into the gaggene at the PstI site at nucleotide 1415 of the gag gene of HIV-1 LAIisolate or the corresponding location of other HIV gag genes.
 20. Thenucleic acid molecule of claim 19 wherein the heterologous nucleic acidinsert comprises from 1 to 4 copies of a DNA sequence selected from thegroup consisting of:(a) 5' GCATTCGACACTAGAAATAGAATAATAGAAGTTGAAAAT 3'(SEQ ID NO:5); and (b) 3' CGTAAGCTGTGATCTTTATCTTATTATCTTCAACTTTTA 5'(SEQ ID NO: 6).
 21. The nucleic acid molecule of claim 1 wherein saidenv gene has been modified to provide a modified env gene product in theHIV retrovirus-like particles in which endogenous anchoring function ofenv has been replaced by a non-retroviral antigenic anchor markersequence operatively connected to the env gene product to anchor saidenv gene product to the HIV retrovirus-like particle, and wherein saidmarker, when presented in the context of the retrovirus-like product, iscapable of generating an immune response specific to said antigenicmarker when the particle is administered to a host.
 22. The nucleic acidmolecule of claim 21 wherein the antigenic anchor sequence comprises aDNA sequence selected from the group consisting of:(a) 5'TGGATCCTGTGGATTCCTTTGCCATATCATGCTTTTTGCTTTG TGTTGTTTTGCTGGGGTTCATCATGTGG3' (SEQ ID NO: 7); and (b) 3'ACCTAGGACACCTAAAGGAAACGGTATAGTACGAAAAACGAAACACAACAAAACGACCCCAAGTAGTACACC 5' (SEQ ID NO: 8).
 23. The nucleic acidmolecule of claim 21 wherein the antigenic anchor sequence includes aDNA sequence selected from the group consisting of:(a) 5'TCAACAGTGGCAAGTTCCCTAGCACTGGCAATCATGATAGCTGTGCTATCTTTTTGGATGTGTTCCAATGGGTCATTGCAG 3' (SEQ ID NO: 9); and(b) 3' AGTTGTCACCGTTCAAGGGATCGTGACCGTTAGTACTATCGACCAGATAGAAAAACCTACACAAGGTTACCCAGTAACGTC 5' (SEQ ID NO: 10).
 24. Thenucleic acid molecule of claim 21 wherein the antigenic anchor sequenceincludes a DNA sequence selected from the group consisting of:(a) 5'TGGATCCTGTGGATTTCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGTTTGCTGGGGTTCATCATGTGGGCCTGCCAAAAAGGCAACGGTGCAACATTTGCATTTGATAGTAAAGAAGAGTGGTGCAGAGA GAAAAAAGAGCAGTGGGAA 3'(SEQ ID NO. 11); and (b) 3' ACCTAGGACACCTAAAGGAAACGGTATAGTACGAAAAACGAAACACAACAAACGACCCCAAGTAGTACACCCGGACGGTTTTTCCGTTGAATCCACGTTGTAAACGTAAACTATCATTTCTTCTCACCACGTCT CTCTTTTTTCTCGTCACCCTT5' (SEQ ID NO:12).